Role of microtubule associated proteins in epithelial apico-basal polarisation and tissue architecture

The cytoskeleton is an integral aspect of many cellular functions and plays an important role in normal tissue development as well as in the pathologies of many diseases. One such function can be seen in the maintenance and formation of apico-basal polarised epithelial cells. Dysregulation of this epithelium causes many developmental diseases and cancers. In fact, 90% of all cancers develop in epithelial cells. As in many systems, microtubules are responsible for the asymmetrical distribution of polarity determinants in the epithelium which is ultimately controlled by the centrosome and microtubule associated proteins. During apico-basal polarisation, the centrosome moves to an apical position where it nucleates microtubules, in kidney epithelium for example. The role and importance of the centrosome in this system is yet to be fully elucidated. Here, the centrosome was removed using the PLK4 inhibitor centrinone B in the MDCKII cell line and apico-basal polarisation was studied in these cells. The centrosome proved essential in the normal organisation of many key microtubule associated proteins in polarising epithelial cells. Amongst these were the relatively new CAMSAP family of proteins where CAMSAP2 and CAMSAP3 lost their non-centrosomal microtubule organising centre localisation in the absence of the centrosome. Markers of polarity were also perturbed in acentrosomal cells suggesting that the centrosome is essential for the formation of apico-basal polarised cells. Furthermore, centrosome removal led to a complete failure of 3D cyst formation further showing evidence of the centrosomes importance in the formation of apico-basally polarised cells. During the differentiation of these apico-basally polarised cells the microtubule end binding protein EB2 is differentially expressed. Here the effect of EB2 overexpression in the MDCKII cell line is studied, analysing many aspects of apico-basal polarisation. EB2 overexpression caused many cellular changes such as mis localisation of the PAR3 polarity marker and a distinct change in junction integrity. Analysis of cell shape suggests these EB2 overexpressing cells became unjammed although it could not be determined if this was due to epithelial-mesenchymal transition or due to the unjamming transition. Cell jamming describes the stability of an epithelial layer with a normal mature layer being jammed with regular cell shape and low motility. Unjamming is a deviation from this solid like state giving more fluid characteristics to the cell layer. Analysis of the centrosome showed increased γ-tubulin and nucleation here although ninein and therefore MT anchorage remained unchanged suggesting that EB2 influences MT nucleation at the centrosome but not anchorage. CRISPR-Cas9 KO of EB2 in MDA-MB-231 cells was attempted in an effort to explore EB2s effect on cell migration, and further distinguish between epithelial-mesenchymal transition and the unjamming transition. This yielded a heterogenous cell line with reduced EB2 expression which caused a significant decrease in cell migration